Fig. 5. The effect of thermal stress on ErbB proteins shares biochemical characteristics with the effects of CI-1033 and GA. (A) The indicated derivatives of 32D cells were treated for 1 h at 37°C with EGF (100 ng/ml), GA (1 µM) or CI-1033 (CI; 10 µM). Alternatively, cells were subjected to heat shock at the indicated temperature for 1 h or left untreated (lanes labeled –). Cell extracts were analyzed by IB and/or IP with the indicated antibodies. (B) The indicated ErbB-2 constructs were expressed in CHO cells along with HA-Ub. Forty-eight hours after transfection, cells were subjected to heat shock at 42°C for 60 min. Cell lysates were analyzed with the indicated antibodies. (C) T47D or T47D-5R cells were subjected to heat shock at 44°C for the indicated time intervals, followed by surface labeling for 40 min on ice with Biotin-X-NHS. Cell extracts were subjected to IP and IB with the indicated antibodies, or with a peroxidase-conjugated streptavidin. Note the reduction in Shc levels in the rightmost lane. (D) COS cells were transfected with expression vectors encoding HA-Ub and one of the four ErbB proteins. Forty-eight hours later, cells were subjected to heat stress for 60 min at 42°C as indicated (+) or left untreated (–). Following lysis, the respective ErbB proteins were analyzed with the indicated antibodies.